PROJECT SUMMARY/ABSTRACT Project: Project 3 Allogeneic hematopoietic cell transplantation (HCT) is frequently curative for otherwise fatal leukemia. However, conventional T cell-replete HCT, using immunosuppressive drugs (IS) alone, is often complicated by graft-versus-host disease (GVHD), which is associated with mortality in 16% of HCT recipients. Quality-of-life is compromised by moderate or severe chronic GVHD. Moreover, prolonged GVHD precludes the use of T cell immunotherapy or vaccination to prevent or treat leukemia that recurs following HCT. T cell depletion is effective for preventing GVHD, but is complicated by slow immune recovery and high rates of opportunistic infection. The objective of this project is to develop an improved HCT strategy to prevent GVHD, rapidly reconstitute pathogen-specific immunity and facilitate future immunotherapeutic approaches to reduce relapse. The research ultimately aims to improve the health and survival of leukemia patients. To improve GVHD-free, relapse-free survival (GRFS), we developed two new HCT approaches: 1) peripheral blood stem cell transplantation (PBSCT) with depletion of nave T cells from the stem cell graft (TND) and 2) PBSCT followed by administration of cyclophosphamide 3-4 days after stem cell infusion (post-transplant Cy; PTCy). In single-arm clinical trials of either TND or PTCy, we observed substantially lower rates of chronic GVHD than is usual among recipients of PBSCT on conventional HCT plans. We now propose in Aim 1 a phase II randomized controlled trial (RCT), comparing PBSCT with TND or PTCy to PBSCT with anti-thymocyte globulin (ATG), the latter a standard GVHD prophylaxis approach, using a flexible, pick-the-winner trial design. Our goal is to select one novel PBSCT strategy to advance to a phase III RCT that will define the best approach for maximizing survival while minimizing relapse, GVHD and dependence on IS. T cell immunotherapy and other immune manipulations delivered in the early post-HCT period have the potential to reduce relapse and would be enabled by identification of bone marrow lymphocyte signatures that presage relapse. Exciting new technologies, including single cell RNA sequencing (scRNA-seq) and TCR deep sequencing (TCR-seq) should facilitate the acquisition of these data and will be evaluated in Aim 2. The specific aims are: Aim 1: To conduct a phase II RCT for patients with acute leukemia comparing two novel strategies (TND and PTCy) to a standard HCT strategy (ATG), aiming to improve GVHD-free, relapse-free survival and avoid prolonged IS after myeloablative allogeneic PBSCT. Aim 2: To evaluate reconstitution and function of peripheral blood and bone marrow immune cell subsets in HCT recipients to determine if lymphocyte signatures are associated with opportunistic infection or AML relapse.